Chemiluminescence-high Performance Liquid Chromatography Research Articles

Novel assay of antibacterial components in manuka honey using lucigenin-chemiluminescence-HPLC

Five components (hydrogen peroxide, methylglyoxal, dihydroxyacetone, fructose and glucose) of New Zealand manuka honey (Leptospermum scoparium) were analyzed using lucigenin chemiluminescence high-performance liquid chromatography (lucigenin-CL-HPLC). We focused on active oxygen species produced from the components in order to easily detect these five components contained in manuka honey. H2O2 and O2− generated from these components were identified by lucigenin-CL and electron spin resonance (ESR), and the bactericidal effect of ROS was confirmed using E. coli. The previously reported assays for Manuka honey components have low specificities and require complicated preprocessing methods. As our results, the detection and identification of these components were possible within 30 min in lucigenin-CL-HPLC system, without any special treatment. It is considered that lucigenin-CL-HPLC is useful for the quality control and the analysis of various honey.

Effect of Curcumin on the Increase in Hepatic or Brain Phosphatidylcholine Hydroperoxide Levels in Mice after Consumption of Excessive Alcohol

Curcumin is a bright yellow compound found in Curcuma longa L., a member of the family Zingiberaceae. In the present study, we determined whether curcumin protects against oxidative stress in liver and brain in mice fed excessive alcohol. BALB/c mice were administered 20% alcohol (16 g/kg body weight) with or without curcumin (0.016% in diet) for 12 weeks. The concentrations of phosphatidylcholine hydroperoxide (PC-OOH) in liver and brain samples were determined using chemiluminescence high-performance liquid chromatography. Mice treated with ethanol and curcumin significantly (P < 0.05) showed a lower hepatic PC-OOH level compared to mice treated with only ethanol. However, there was no significant difference in the brain PC-OOH level among all mice. Our finding indicates that the dosage of alcohol might increase the lipid peroxide level of liver but not of brain, and daily curcumin consumption might be protective for liver against alcohol-related oxidative stress in mice.

Ion-trap tandem mass spectrometric analysis of squalene monohydroperoxide isomers in sunlight-exposed human skin

We previously discovered that squalene monohydroperoxide (SQ-OOH) was produced on human forehead skin and suggested that skin squalene (SQ) may be the principal target lipid for oxidative stress (e.g., sunlight exposure). Because of its six double bonds, SQ peroxidation can yield various positional hydroperoxide isomers. However, the structural characterization of skin SQ-OOH isomers has never been reported. Here, we prepared pure SQ-OOH isomers and developed an analytical method for SQ-OOH isomers using a quadrupole/linear ion-trap mass spectrometer (QTRAP) MS/MS system. Collision-induced dissociation produced specific fragment ions for each SQ-OOH isomer, which permitted discrimination between SQ-OOH isomers by multiple reaction monitoring (MRM). When lipid extract from human forehead skin was subjected to LC-MS/MS with MRM, individual SQ-OOH isomers could be separated and detected with a sensitivity of 0.05 ng/injection. The total concentration of SQ-OOH isomers in forehead skin was approximately 956 microg/g skin lipids, but it increased up to 2,760 microg/g skin lipids after 3 h of sunlight exposure. The LC-MS/MS method was useful for investigating the peroxidation mechanisms of SQ as well as SQ-OOH-mediated skin disorders.

Determination of propylthiouracil and methylthiouracil in human serum using high-performance liquid chromatography with chemiluminescence detection

Based on the sensitizing effect of formaldehyde on the chemiluminescence (CL) reaction of propylthiouracil (PTU) and methylthiouracil (MTU) with acidic potassium permanganate and the combination technique of high-performance liquid chromatography (HPLC), a sensitive, selective and simple post-column CL detection method for determining PTU and MTU is described. The optimal conditions for the CL detection and HPLC separation were carried out. The linear ranges were 0.1–20 μg mL −1 for MTU and 0.1–10 μg mL −1 for PTU, the detection limits were 0.03 μg mL −1 for PTU, 0.03 μg mL −1 for MTU and the quantification limits were 0.1 μg mL −1 for PTU, 0.1 μg mL −1 for MTU. The method has been satisfactorily applied for the determination of MTU and PTU in human serum samples.

Post-column detection of benzenediols and 1,2,4-benzenetriol based on acidic potassium permanganate chemiluminescence

Based on the sensitizing effect of formic acid on the chemiluminescence (CL) reaction of polyhydroxylbenzenes with acidified potassium permanganate and the combination technique of high-performance liquid chromatography (HPLC), a sensitive, selective and simple post-column CL detection method for simultaneously determining catechol, resorcinol, hydroquinone and 1,2,4-benzenetriol is described. The optimal conditions for the CL detection and HPLC separation were carried out. The linear ranges were: 6.0 × 10 −3–1.5 mg/L for hydroquinone, 8.0 × 10 −3–1.5 mg/L for 1,2,4-benzenetriol, 1.0 × 10 −2–2.0 mg/L for resorcinol and 1.0 × 10 −2–2.5 mg/L for catechol, respectively. The detection limits are: 3.2 × 10 −3 mg/L for hydroquinone, 3.9 × 10 −3 mg/L for 1,2,4-benzenetriol, 4.7 × 10 −3 mg/L for resorcinol and 5.2 × 10 −3 mg/L for catechol, respectively. Combining with solid phase extraction, the proposed method has been successfully applied to the determination of the polyhydroxylbenzenes in river water. The recoveries for three benzentriols were 92.1–95.4% and 82.0% for 1,2,4-benzenetriol, respectively.

Detection of synthetic corticosteroids in bovine urine by chemiluminescence high‐performance liquid chromatography

The development of a black market of chemical cocktails for illegal growth promotion in food-producing animals includes substances that are potentially dangerous for human health, such as synthetic corticosteroids. The potential presence of these residues in food makes it necessary to develop rapid and sensitive analytical methodologies to detect such substances, preferably in live animals before they arrive at the market. A chemiluminescence (CL) detection method for the determination of four synthetic corticosteroids (prednisolone, betamethasone, dexamethasone and flumethasone) in bovine urine has been developed. The proposed system, which does not need any derivatization procedure, offers an easy method well suited for routine research. Urine samples were homogenized with methanol:water (50:50, v/v) and centrifuged. The upper layer was collected and Strata X cartridges were used for cleaning up. The purified residues were evaporated to dryness and then redissolved in the mobile phase. Analysis of the extracts was performed using high-performance liquid chromatography with chemiluminescence detection, employing luminol as the CL reagent. The recovery curves, obtained at four spiking levels (different for each corticosteroid), showed that recoveries of at least 70% could be obtained for urine. The chemiluminescence detection procedure afforded satisfactory results with respect to sensitivity and the LOD and LOQ, taken as the first point of the regression curve, ranged from 4 ppb to 65 ppb. The maximum mean RSD was below 13% and below 15% for intra- and inter-day assay, respectively, in all cases.

Increase of serum phosphatidylcholine hydroperoxide dependent on glycemic control in type 2 diabetic patients

In order to clarify the relationship between serum phosphatidylcholine hydroperoxide (PCOOH) levels and blood glucose control in type 2 diabetes patients (DM), DM ( n=61) and normal control ( n=11) were enrolled. High-density lipoprotein (HDL) was separated from serum by the addition of sodium phosphotungstate and magnesium chloride, and the precipitated fraction was prepared as non-HDL. Phospholipids were extracted from whole serum, non-HDL and HDL to estimate PCOOH level with chemiluminescence high performance liquid chromatography (CL-HPLC). PCOOH level (nmol/l, mean±S.D.) was higher in DM than in control (33.1±9.5 vs. 23.0±8.2 for serum; P<0.01, 17.0±5.5 vs. 10.6±3.8 for non-HDL; P<0.01, and 16.1±6.3 vs. 12.3±5.5 for HDL; not significant, respectively). DM was divided into five groups according to hemoglobin A 1c (HbA 1c) levels (%): (1) less than 6, (2) 6–6.4, (3) 6.5–6.9 (4) 7.0–7.4, and (5) over than 7.5. Increase of PCOOH levels was dependent on HbA 1c. We concluded that (1) serum and non-HDL PCOOH increased in DM, (2) the level was strongly correlated with diabetic control, and (3) approximately a half amount of serum PCOOH was present in HDL of both control and DM.

Dietary supplementation of N-3 fatty acids and hydroperoxide levels in rat retinas

Docosahexaenoic acid (DHA) plays an important role in visual and neural development in mammals. In the present study, effect of dietary supplementation with n-3 fatty acids, primarily docosahexaenoic acid (DHA) with high purity, on the fatty acid composition of photoreceptor cells of young rats (fed from 4 weeks) was investigated. DHA in rod outer segment (ROS) membranes was significantly increased in the group of high DHA feeding (9.69% total energy). Other n-3 fatty acids (α-linolenic acid (ALA) and eicosapentaenoic acid (EPA)) included in the diets with DHA (0.95%∼5.63% total energy) also significantly increased the proportion of DHA compared with the linoleic acid diet groups. However, the proportions of arachidonic acid (ARA) and other long chain n-6 fatty acids (22:4n6 and 22:5n6) were suppressed in these n-3 fatty acids-fed groups. Phospholipid hydroperoxides in ROS membranes were determined using a highly sensitive analytical technique, chemiluminescence-high performance liquid chromatography (CL-HPLC). There was no increasing tendency in the hydroperoxide levels of ROS membranes containing high content of DHA, and phosphatidylethanolamine hydroperoxide (PEOOH) was much lower than phosphatidylcholine hydroperoxide (PCOOH) under normal light conditions, which implies that DHA supplementation does not much affect the peroxidizability of ROS membranes in vivo. But UV irradiation on separated ROS membranes accelerated the formation of phospholipid hydroperoxides in high DHA feeding rats, and PEOOH was produced more efficiently than PCOOH in vitro.

The role of lipid peroxidation on gastric mucosal lesions induced by water-immersion-restraint stress in rats.

This study was designed to determine the effect of water-immersion-restraint stress (WIRS) and pretreatment by reduced glutathione on both the production of gastric mucosal lesions and the content of gastric mucosal phosphatidylcholine hydroperoxide (PCOOH). Sprague-Dawley rats were divided into control and treatment groups (treated with reduced glutathione before stress). After graded durations of WIRS, the macroscopic ulcer index (UI) was measured and the PCOOH content was examined by a chemiluminescence-high performance liquid chromatography assay. The UI in the control group increased significantly in a time-dependent fashion. Elevation of the PCOOH level was observed in combination with the UI for up to 2h of WIRS, but then showed a declining tendency. Pretreatment with reduced glutathione significantly lowered both the UI and the PCOOH level. Lipid peroxidation is probably involved in the pathogenesis of gastric injury induced by WIRS at least in the early phases. However, in the late phases, other mechanisms causing gastric mucosal lesions may also be involved. We therefore believe this study to be the first to accurately and quantitatively assess the degree of lipid peroxidation in the gastric mucosa as a result of stress.

Determination of dexamethasone in bovine liver by chemiluminescence high-performance liquid chromatography.

A new method for the determination of dexamethasone (9alpha-fluoro-11beta,17alpha,21-trihydroxy-16alpha -methylpregna-1, 4-diene-3,20-dione) in bovine liver was developed. This new liquid-liquid extraction method comprises the addition of sodium hydroxide to the tissue sample followed by extraction with ethyl acetate. After centrifugation, the extract is evaporated to dryness and the residue dissolved in acetonitrile. The cleaning of the fat is performed with n-hexane, and the acetonitrile layer is evaporated. Analysis of the extracts is performed using high-performance liquid chromatography with chemiluminescence detection employing luminol as CL reagent. A series of recovery curves performed at spiking levels of 50, 30, 10, 5, and 2.5 ppb show that at least 80% of DEX can be recovered from liver and that the chemiluminescence detection yields satisfactory results with respect to sensitivity (LOD 0.2 ppb), reproducibility (CV% 10.7) and repeatability (CV% 6.2-8.9).

Development of Electrogenerated Chemiluminescence HPLC for Ofloxacin Using Tris (2, 2′-bipyridine) ruthenium (II) and Its Application to Residual Analysis in Chicken Tissues

An electrogenerated chemiluminescence high performance liquid chromatography (ECL-HPLC) for ofloxacin (OFLX) using tris (2, 2′-bipyridine) ruthenium (II) was developed and applied for residue analysis in chicken tissues. OFLX was extracted from tissues by homogenizing with 0.1mol/L phosphate buffer (pH 12)-acetonitrile (1:3, v/v) and centrifuging the mixture. The supernatant was shaken with n-hexane saturated with acetonitrile. After having been centrifuged, the lower layer was evaporated to dryness under reduced pressure and the residue was dissolved in acetonitrile-0.05mol/L NaH2PO4 (pH 2.4) (20:80, v/v) and analyzed by ECL-HPLC. The mean recoveries of OFLX from chicken tissues were 66-102%. The detection limit of this method was 10ng/g (0.01ppm) of OFLX. The correlation between ECL-HPLC and FL-HPLC was excellent.

Chemiluminescence High Performance Liquid Chromatography of Corticosteroids and p-Nitrophenacyl Esters Based on the Luminol Reaction

Reducing agents, especially compounds having an α-hydroxycarbonyl group, give an intense chemiluminescence with luminol in the presence of catalyst in alkaline solution. Based on this reaction, the chemiluminescence detection of corticosteroids and p-nitrophenacyl esters of carboxylic acids has been developed. These compounds were separated by high performance liquid chromatography on a reversed phase column (Prodigy ODS) for corticosteroids with 30% acetonitrile and for p-nitrophenacyl esters with 50% acetonitrile as eluent, and detected by the chemiluminescence reaction with luminol-sodium hydroxide solution containing the catalyst as the post-column reagent. Hexacyanoferrate (III) and hexacyanoferrate (II) were used as catalyst of the reaction. The detection limits of cortisone and p-nitrophenacyl hippurate were 1.1 pmol and 0.8 pmol.

Phosphatidylcholine hydroperoxide in gastric mucosal lesions induced by HCl, NaOH or ethanol: Detection using luminol chemiluminescence-high performance liquid chromatography: [formula omitted], I. Sasaki, H. Naito, T. Tsuchiya, S. Matsuno. 1st Dept. of Surgery, School of Medicine, Tohoku University, Sendai, Japan

Phosphatidylcholine hydroperoxide in gastric mucosal lesions induced by HCl, NaOH or ethanol: Detection using luminol chemiluminescence-high performance liquid chromatography: [formula omitted], I. Sasaki, H. Naito, T. Tsuchiya, S. Matsuno. 1st Dept. of Surgery, School of Medicine, Tohoku University, Sendai, Japan

Peroxidation in Human Skin and Its Prevention

Cosmetics and quasi-drugs will play new important roles in the aging and stressful society. We have proposed 3 clinical concepts, i.e., anti-drying, anti-UV radiation and anti-oxidation to prevent the skin aging. Especially, the importance of anti-oxidation is discussed in this paper. The peroxidation of human skin surface lipids has been investigated by a CL-HPLC (chemiluminescence-high performance liquid chromatography) system. Squalene monohydroperoxide was produced at the forehead and the scalp as well as in the dandruff under daily life conditions in most of the cases. Therefore it was considered that squalene was the first target lipid on a human skin surface by an oxidative stress. In order to confirm the effect of hydroperoxides of the skin on cultured cells and the living skin equivalent, an authentic sample was prepared. We have also clarified the reaction rate constants of the skin surface lipids with singlet oxygen and free radicals. And we have also confirmed that the application of cosmetics and the ingestion of aliments containing antioxidants had a preventive effect against the lipid peroxidation on skin surface.

Behavior of Lipid Hydroperoxides During Mashing

Lipid hydroperoxides such as trilinolein hydroperoxides in barley, malt, and mash were analyzed using a chemiluminescence high-performance liquid chromatography method. During mashing in a laborato...

DETERMINATION OF FATTY ACID HYDROPEROXIDES PRODUCED DURING THE PRODUCTION OF WORT

Linoleic and linolenic acid hydroperoxides in malt, mash, or wort were determined with high sensitivity and high selectivity by the chemiluminescence-high performance liquid chromatography (CL-HPLC) method using isoluminol-microperoxidase solution as a luminescing reagent. The determination limit of this method for both hydroperoxides was 0.1 μM in mash or wort. During the mashing in a laboratory mash bath, the hydroperoxides started to increase just after mashing-in, reached a maximum at 65°C, and then decreased. Though the hydroperoxides were detected in mash just before the lautering in a pilot scale brewing, they disappeared during the lautering and could not be detected during the subsequent stages of wort production. Therefore, it was thought that the mashing process is the most important of the lipid oxidation reactions during wort production. It is also expected that the CL-HPLC method can give useful information on lipid oxidation mechanisms during wort production.

Detection of Squarene Hydroperoxide

Squalene hydroperoxide present in human skin surface lipids was examined by a chemiluminescence high performance liquid chromatography (CL-HPLC) system. A mixture of cytochrome c and luminol served as the hydroperoxide-specific luminescence reagent for post-column detection in reversed phase HPLC. Squalene hydroperoxide was previously detected in skin surface lipids of the human forehead following exposure to the sun. Therefore, in this study, squalene monohydroperoxide was prepared by dye-sensitized photooxidation and its chemical structure was determined by 1H-NMR, 13C-NMR, and FAB-MS. The detection limit of squalene monohydroperoxide in CL-HPLC system was as low as 1 pmol. Skin surface lipids from forehead, scalp, and dandruff were also studied. By exposure to sun light or after washing with shampoo, squalene monohydroperoxide could be detected in nearly all samples examined. Squalene may thus be considered the first target lipid on human skin surface that incurs oxidative stress by sun light exposure, etc.

ヒト表皮における脂質過酸化反応

The peroxidation of human skin surface lipids was investigated by a CL-HPLC (chemiluminescence-high performance liquid chromatography) system. Squalene mono-hydroperoxide was produced at the forehead and the scalp as well as in the dandruff under daily life conditions in most of the cases. Therefore it was considered that squalene was the first target lipid on a human skin surface by an oxidative stress. In order to confirm the effect of squalene monohydroperoxide on proliferation of cultured cells and TESTSKINTM (Living Skin Equivalent), an authentic squalene mono-hydroperoxide was prepared by a dye-sensitized photooxidation and its chemical structure was identified by 1H-NMR, 13C-NMR and FAB-MS. Detection limit of squalene mono-hydroperoxide in CL-HPLC system was as little as 1 pmol. We also found that cosmetics containing antioxidants had a preventive effect against the lipid peroxidation on a Human Skin Surface.

The inverted hexagonal phase is more sensitive to hydroperoxidation than the multilamellar phase in phosphatidylcholine and phosphatidylethanolamine aqueous dispersions

The effect of phase behaviour (hexagonal II phase and lamellar phase) on the peroxidation of membrane phospholipids has been investigated in dilinoleoyl phosphatidylcholine (DLPC)/dilinoleoyl phosphatidylethanolamine (DLPE) aqueous dispersions. Peroxidation was initiated with a water-soluble radical inducer 2,2′-azobis (2-amidino-propane) dihydrochloride (AAPN). The phospholipid morphology was monitored by 31P-nuclear magnetic resonance (NMR). Phospholipid hydroperoxides (PCOOH and PEOOH) were determined by chemiluminescence high-performance liquid chromatography (CL—HPLC). In pH-induced phase transition systems, DLPE in the bilayer state was much less oxidized than in the hexagonal II state. In composition-induced phase transition systems, the formation of total hydroperoxides and the consumption of α-tocopherol in the hexagonal II phase were greater than in the bilayer phase. These data suggest that the hexagonal II phase is more sensitive to hydroperoxidation than the bilayer phase in phospholipid aqueous dispersions.

Determination of Human Skin Surface Lipid Peroxides by Chemiluminescence-HPLC

The peroxidation of human skin surface lipids was investigated by the CL-HPLC (chemiluminescence-high performance liquid chromatography) system, using a reversed phased HPLC column and hydroperoxide-specific luminescent reagent consisting of cytochrome c and luminol. Squalene hydroperoxide was detected as the most prominent chemiluminescent peak in oxidized skin surface lipids, and the retention time was identical with that of hydroperoxide of the autoxidized squalene. The chemiluminescent peak disappeared by sodium borohydride treatment. Skin surface lipids oxidized by natural exposure to sunlight were analyzed quntitatively and squalene hydroperoxide formation was confirmed.